Insulin-degrading enzyme: stable expression of the human complementary DNA, characterization of its protein product, and chromosomal mapping of the human and mouse genes.
We have recently described the isolation of a cDNA encoding an enzyme thought to be involved in the degradation of insulin by insulin-responsive tissues. This enzyme, referred to as insulin-degrading enzyme (IDE), is a cytosolic proteinase of 110,000 mol wt which shares structural and functional homology with bacterial protease III. The enzyme may function in the termination of the insulin response. We report here the mapping of the human and mouse IDE genes to human chromosome 10 and mouse chromosome 19, respectively, and evidence for the existence of a single complex IDE gene. We also describe the stable transfection of Chinese hamster ovary cells with a plasmid containing the IDE cDNA under the transcriptional control of the SR alpha promoter. The recombinant protein synthesized by these cells is indistinguishable from the isolated human enzyme in both its size and immunoreactivity and degrades insulin with a specific activity similar to that of the purified proteinase. Overexpression of IDE using this system should allow for a functional test of the role of IDE in insulin action. In addition, expression of various site-directed mutants of IDE will aid in identifying the residues of IDE and protease III that are essential to the activity of this unique family of proteinases.
Affholter
J A
JA
Department of Pharmacology, Howard Hughes Medical Institute, Stanford University School of Medicine, California 94305.
Hsieh
C L
CL
Francke
U
U
Roth
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007339:Insulysin / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D007328:Insulin; D011994:Recombinant Proteins; D004247:DNA; D007339:Insulysin